High-Performance Liquid Chromatography (HPLC) is one of the most widely used separation techniques (J. G. Dorsey et al., Anal. Chem. 1998, 70: 591–644); and its applications range from industrial preparation to trace level detection. The popularity of HPLC stems from its ability to separate a large variety of analytes, including organics, inorganics and biomaterials with low to high molecular weights, and with different degrees of polarity, hydrophobicity, acidity, and ionization (J. Swadesh, HPLC. Practical and Industrial Applications, CRC Press, Boca Raton, 2001; J. S. Fritz, Ion Chromatography, Wiley-VCH, Weinheim, 2000). The development of highly sophisticated HPLC procedures has provided a sensitivity that allows researchers to separate enantiomers (A. Satinder, Chiral Separations by Chromatography, Oxford University Press, Washington, D.C., 2000) and even isotopes; to detect mutations in DNA fragments; and to achieve resolution of very similar polypeptides that differ by a single amino acid residue. The vast majority of biomedical and environmental analyses are currently performed by HPLC; and in the pharmaceutical industry, where it is the premier analytical technique, HPLC is used in all phases of drug discovery, development, and quality control.
Chromatography may be defined as the separation of analyte molecules based on their differing affinities for distinct phases in relative motion. Essentially, in HPLC, components of a sample mixture are carried through a solid stationary phase by the flow of a liquid mobile phase, that is pumped under controlled conditions of high pressure. The stationary phase, which is composed of a packed bed of finely divided beads or particles contained within a chromatography column, acts as a retentive media and differentially retards the migration of the analytes through the column so that the individual components of the mixture are gradually separated from one another and ultimately exit the column at different time points of the chromatographic run.
Successful chromatography requires a proper balance of the intermolecular forces between the analyte, the mobile phase, and the stationary phase. In HPLC, both the mobile phase and stationary phase may be varied to alter the interaction mechanism(s). The important criteria to consider for HPLC method development are resolution, sensitivity, precision, accuracy, limit of detection, linearity, reproducibility, time of analysis and robustness of the method. The quality of the column itself, and its performance over time, contribute in important ways to each of these criteria.
In spite of recent advances in the development of alternative stationary phases such as zirconia, alumina, titania, and polymer-based packings (K. K. Unger, Packings and Stationary Phases in Chromatographic Techniques, Marcel Dekker, New York, 1990), microparticulate silica is by far the most commonly employed chromatographic support in HPLC. This is mainly due to its versatility, high derivatization potential, high column efficiency, and easily controlled particle size and porosity. Most of the silica-based packings in use today are bonded phases that are formed by covalently attaching organic molecules with specific properties to silanol groups (Si—OH) present on the silica surface. Of these silica gel-based support materials, reversed-phase (i.e., weakly polar or non-polar) bonded packings are generally preferred because of their high resolution power, separation efficiency, and mechanical stability.
Although superior to most other available packings, silica-based bonded phases remain imperfect supports for reversed-phase HPLC, especially in the analysis of basic substances. The derivatization process that produces the bonded phase is rarely fully complete and generally leaves unreacted a significant number of silanol groups on the silica surface. The presence of these residual weakly acidic silanols leads to irreversible adsorption of basic solutes, high peak tailing, and a strong dependence of retention times on solute concentrations (H. Engelhardt et al., J. Chromatogr. 1988, 458: 79–92). Separations at high pH (i.e., pH 9 or greater) appear, therefore, attractive for certain basic analytes since, under these conditions, (1) such analytes would be in their free-base form, and (2) the unreacted weakly acidic silanol groups would be totally ionized, a situation where irreversible and potentially deleterious electrostatic interactions between solute molecules and stationary phase would be minimized. Furthermore, operating at a pH well above the pKa value of basic compounds should also allow more reproducible separations, since retention times changes due to the formation of ionized forms would not take place (J. J. Kirkland et al., J. Chromatogr. 1997, 762: 97–112; J. J. Kirkland et al., J. Chromatogr. A, 1998, 797: 111–120). However, using reversed-phase HPLC columns under intermediate to high pH aqueous conditions, especially at elevated temperatures, is known to result in rapid loss of column performance and in reduced column lifetimes due to deterioration of the packing material, largely through solubilization of the silica support (J. J. Kirkland et al., J. Chromatogr. A, 1995, 691: 3–19; C. S. Horvath et al., Anal. Chem. 1977, 49: 142–154; A. Wehrli et al., J. Chromatogr. 1978, 149: 199–210).
Several studies have been carried out to devise ways to extend the lifetime of reversed-phase HPLC columns. Thus, the use of untreated silica pre-columns that partially saturate the mobile phase with dissolved silica, thereby reducing or precluding solubilization of the packing material in the analytical column, has been found to significantly improve the longevity of a column (J. G. Atwood et al., J. Chromatogr. 1979, 171: 109–115). Addition to the mobile phase of a large variety of certain additives and modifiers, such as organics, salts and detergents (L. C. Sander and S. A. Wise, CRC Crit. Rev. Anal. Chem. 1987, 18: 299), that interact with the analytes and/or the stationary phase, has been reported to increase the lifetime of HPLC columns by, for example, preventing irreversible adsorption of basic solutes to the reversed-phase support and/or by decreasing the solubility of microparticulate silica at intermediate to high pH.
Chemical approaches have also been developed to extend the lifetime of reversed-phase HPLC columns by shielding, eliminating, or reducing the number of residual silanol groups on the silica surface (K. K. Unger, Porous Silica, Elsevier, Amsterdam, N.Y., 1976; J. Kohler and J. J. Kirkland, J. Chromatogr. 1987, 385: 125–150; D. B. Marshall et al., J. Chromatogr. 1986, 361: 71–82; J. G. Dorsey and W. T. Cooper, Anal Chem. 1994, 66: 857A–867A). Work has, for example, been directed at increasing the efficiency of the derivatization reaction in order to produce more densely bonded silica supports with a smaller number of unreacted silanols. Silanol groups have also been partially removed by curing, a condensation reaction of adjacent silanols (T. G. Waddell et al., J. Am. Chem. Soc. 1981, 103: 5303–5307), or by end-capping, a chemical process in which relatively small reagents (such as activated silanes) are reacted with the remaining silanols (G. B. Cox, J. Chromatogr. A, 1993, 656: 353–367; J. J. Kirkland et al., J. Chromatogr. A, 1997, 762: 97–112). Bonding of bidendate silanes or bulky silanes (J. J. Kirkland et al., Anal. Chem. 1989, 61: 2–11) with tentacle-like chains (M. Ashri-Khorassani, et al., Anal. Chem. 1988, 60: 1529–1533) has been shown to produce stationary phases with improved stability properties as compared with the commonly used monofunctional packings. Similarly, polymeric (T. Darling et al., J. Chromatogr. 1977, 131: 383–390; A. J. Alpert and F. E. Regnier, J. Chromatogr. 1979, 185: 375–392) and horizontally polymerized silanes (G. Schomburg et al., J. Chromatogr. 1983, 282: 27–39; M. J. Wirth and H. O. Fatunmbi, Anal. Chem. 1993, 65: 822–826) have been found to exhibit some stability in high pH mobile phases. In a slightly different approach, treatment of silica particles with metal oxides or hydroxides, which results in the formation of a protective layer over the silica, has also been demonstrated to increase the lifetime of the stationary phase thus obtained (see, for example, U.S. Pat. No. 4,600,646).
Although most of the modifications of the silica surface generate chromatographic supports with improved stability properties at medium to high pH, several of the chemical reactions cannot easily be applied to many of the currently commercially available columns.
Therefore, a need continues to exist for improved strategies for extending the lifetime of silica gel-based reversed-phase HPLC columns. There is a particular need for approaches that are widely applicable, do not involve chemical modifications of the stationary phase, increase the retention of analytes in the column, and yet preserve the integrity of the HPLC column.
Such strategies would for example be useful in the case of a particular superficially porous silica-based packing material that has been reported to show great performance for the separation of large molecules. This packing material, which is composed of particles made of a solid silica core and a macroporous shell, cannot practically be used at medium pH (i.e., 6–8) and at high temperature (i.e., >50° C.) since these conditions cause a dramatic shortening of its lifetime. Methods to increase the longevity of this packing material under the above conditions, which are typical for the analysis of some large biomolecules such as DNA, oligonucleotides, and some proteins, would considerably widen the range of applications of this chromatographic support. Furthermore, in the case of DNA fragments, it may be desired that the retention of the fragments take place in the order of the fragments' length regardless of their composition. A reverse order is sometimes observed due to interactions with silanol residues on the silica surface. Methods that would reduce or eliminate this problem and thus allow DNA fragment sizing are therefore highly desirable.